Gap junction channels are oligomers of connexins encoded by a gene family, which in mammals has 12 members classified phylogenetically into Group I and Group II. Different connexins form gap junctions that differ markedly in their unitary conductances, steady-state and kinetic responses to voltage, and effects of phosphorylation. Gap junctions are believed to be formed of two hemichannels in series, each with its own gating mechanisms. However, heterotypic junctions can display unexpected properties, and in some cases one hemichannel appears to modify the gating properties of the other. Recently, we identified a charge complex in two closely related Group I connexins, Cx32 and Cx26, that appears to be part of the transjunctional voltage, Vj, sensor and we proposed a molecular mechanism of gating in these and other Group I connexins. In this project we focus on Group II connexins, which have characteristic differences in sequence. Specific Aim 1 is to determine differences in gating properties of Group II connexins and from sequence comparisons identify sites where changes in charge appear likely to affect voltage gating. We will then test gating hypotheses in Specific Aim 2 by site directed mutagenesis and domain exchange and determine how hemichannel interactions influence voltage gating. We will establish whether our voltage gating model developed for Group I connexins is applicable to Group II connexins and whether common structural motifs mediate voltage gating in the connexin family. We find that Cx46, which has the capacity to form functional hemichannels, can be readily examined at the single channel level in both cell-attached and excised membrane patch configurations. Specific Aim 3 is to examine ion selectivity and kinetics of gating in these hemichannels. In Specific Aim 4 we will examine the molecular determinants of these properties by molecular genetic methods and in Specific Aim 5 we will attempt to transfer the capacity of Cx46 to function as an isolated hemichannel to other connexins. If successful this approach will allow selectivity and voltage gating to be addressed directly in these other connexins as well. Specific Aim 6 is to examine the role of phosphorylation in modulation of gating by taking advantage of the well documented actions of serine and tyrosine kinases on the C-terminus of Cx43. By domain exchange and point mutagenesis we will explore the molecular basis whereby alterations in channel gating are produced by phosphorylation. The proposed studies should greatly increase our knowledge of voltage gating and permeation and provide functional explanations for the specificity of connexin expression and a mechanistic basis for interpretations of genetic diseases associated with gap junctions.